Our current work focuses primarily on understanding the differences in microtubule organization between muscles of normal mice (WT) and of mdx mice, a model for Duchenne muscular dystrophy (DMD). Normal mouse muscles have a periodic grid-like microtubule network, whereas mdx mouse muscles have a disordered, denser network. The software TeDT, developed in the Light Imaging Section for the analysis of microtubule directionality, is an essential tool in the quantitative assessment of such differences in microtubule organization (Liu et al., 2014). Results obtained in previous years demonstrated that the differences in microtubule orientation can be observed as soon as microtubules start growing from the nucleating Golgi elements. Thus muscle microtubules grow as if Golgi elements themselves, or the nucleating molecules anchored to the Golgi elements, had a specific orientation, disturbed in mdx muscles. Conventional and super-resolution microscopy have been used to investigate the orientation of the Golgi elements. Labeling of the Golgi elements with two antibodies, one for the cis-Golgi protein GM130, the other one for the trans-Golgi protein TGN38 allowed us to determine the orientation of each Golgi element. Plotting Golgi directionality with the software TeDT indeed shows differences between WT and mdx mouse muscles, confirming that differences in microtubule organization are sealed at an early stage of their formation. We have also carried out RNA-seq analysis of three different mouse muscles (FDB, EDL, and soleus) at two different ages, 2 and 5 months, from both WT and mdx mice. The goal was a comparison of RNA changes in the mdx mouse muscles compared to human DMD muscles (Khairallah et al. 2012, Sci Signal, 5, ra56)) with special attention to tubulins, the constituents of microtubules, and microtubule-associated proteins. The two ages were selected based on the report in the same paper that microtubules are implicated in the mdx pathology at 5 but not at 2 months of age. The results confirm that several tubulin mRNAs are differentially expressed in mdx compared to WT muscles. However, they also point to differences between human and mouse tubulin isoform distribution. Although several mRNAs are differentially expressed in 2 and 5 mo-old mice, this is not the case for tubulin mRNAs. Thus, the difference in response between these ages must be searched in other parts of the pathways affected. After analysis of the results is complete we will further investigate the role of specific molecules hypothesized to play a role in the disorganization of the mdx network. Our research over the past year has focused on the beta-tubulin isoform encoded by the gene tubb6. The full name of the protein is tubulin beta6 class V. For the sake of simplicity we refer to it as TUBB6. There are two motives to our interest for this tubulin: first, its mRNA is the most highly increased when comparing DMD to normal human muscle transcriptome; second, overexpression of this ubiquitous, minor beta-tubulin isoform modifies the microtubule network and is toxic to proliferating cells (Bhattacharya et al. 2011, Mol Biol Cell, 22, 1025-34). We decided to pursue two goals: on one hand investigate the effects of overexpressing TUBB6 in normal mouse muscle; and on the other hand explore what happens when TUBB6 is decreased in mdx muscle. Both avenues have been successful and suggest TUBB6 as disease modifier for the mdx mouse and potentially for DMD. Overexpression of a GFP construct of TUBB6 led to a distorted, dense microtubule network in WT mouse muscle. In comparison, overexpression of the same GFP construct of another beta tubulin, TUBB5, caused no modification of the microtubule network. When TUBB6 was decreased or suppressed in mdx muscle by shRNA treatment, the fibers expressing the shRNA --but not a scrambled shRNA or an RNA against TUBB5-- showed a nearly normal microtubule network. Thus, manipulating the level of TUBB6 allows us to modify the organization of muscle microtubules, regardless of the presence or absence of dystrophin, the protein that is lacking in DMD and mdx and that we thought necessary for the guidance and proper organization of microtubules.